Chemoenzymatic synthesis employs both chemical and biocatalytic steps in a reaction sequence. The biocatalytic transformations convert one organic compound to another by use of enzymes, either isolated or as part of biological systems.
Enzymes derived from biological systems (for example, from a microorganism or an animal organ) have been particularly useful in the resolution of racemic compounds. In these systems, a chiral compound composed of two enantiomers is used as the substrate for the enzyme. The enzyme specifically recognizes and favors only one of the enantiomers as the substrate for the enzymatic reaction. The stereospecificity of the enzyme optimally affords a product mixture having a 50% conversion to a single enantiomer product and 50% recovered starting material of opposite configuration.
The product mixture can be analyzed for enantioselectivity by numerous methods. The optical purity of the products defines the degree of enantioselectivity of an enzymatic resolution and can be expressed as the "E" value, a directly proportional measure of the R to S reactivity rate ratio. Because the "E" value is independent of conversion, it is particularly useful in evaluating kinetic resolutions, as described in Chen, C. S, et al, J. A Chem. Soc. 1982, 104, p. 7249. These "E" values are determined by the optical purity of both the product and recovered starting material, with higher optical purities affording higher "E" values.
For present purposes, an "enantiomerically enriched" compound is defined as having an enantiomeric excess ("ee") of greater than about 80%.+-.2%. The enantiomerically enriched product is desirable because it can be further converted into various enantiomerically enriched compounds. For example, single enantiomeric compounds are useful in the synthesis of pharmaceutical compounds where one enantiomeric form of the compound may be pharmaceutically active and the other enantiomeric form may be inactive or even detrimental.
There are many different methods that are used, including biocatalysis, to prepare enantiomerically enriched compounds. Hydrolase enzymes have been employed with success for the stereospecific preparations of alcohols and esters using several reaction variants. The conventional enzymatic hydrolysis reaction is normally performed in aqueous media with pH control and entails hydrolysis of a chiral ester substrate to afford the corresponding acid and alcohol products. Enzymatic transesterification commonly also refers to the use of a chiral ester substrate, but the products are a second ester and the alcohol portion of the substrate from the reaction of the acylated enzyme with an alcohol rather than water. The enzymatic esterification of a chiral substrate alcohol is quite different, being a synthetic rather than a hydrolytic process with respect to the substrate.
The enzymatic esterification of an alcohol derivative in an organic solvent relies on the absence of water to reverse an equilibrium which normally favors ester hydrolysis rather than synthesis, as diagramed in the reaction sequence shown below. An achiral acyl donor acylates the enzyme in the first step (I). In the second step (II) the acyl-enzyme reacts with the desired chiral substrate alcohol to form a corresponding enantiomerically enriched product ester, an unreacted enantiomerically enriched alcohol of the opposite configuration, and free enzyme. ##STR1##
The choice of the X radical of the acyl donor is significant in assisting the equilibrium reversal of the reaction. If the X radical is a simple alcohol, the radical can concrete favorably with the desired alcohol for the acyl-enzyme complex (k.sub.-1 k.sub.2) and thus slow the desired reaction immensely. To improve the reaction rate, either the nucleophilicity of the alcohol released upon enzyme acylation can be reduced or else the first step of the reaction can be made irreversible. For example, vinyl esters are preferred as acyl donors because the alcohol portion, upon release from the ester, completely isomerizes to the corresponding aldehyde, effectively shutting down k.sub.-1.
In many cases, the enzymatic esterification reaction of a chiral substrate has been found to offer significant operational advantages over the corresponding enzymatic hydrolysis. Besides being a step shorter (the preparation of the racemic ester is unnecessary), pH control of the reaction mixture is avoided. In the enzymatic esterification, filtration to remove the enzyme followed by solvent removal affords the products in nearly quantitative yield without requiring the extractive isolation procedure usually necessary in the enzymatic hydrolysis reaction. Further, reuse of the enzyme is possible in the enzymatic esterification, whereas in the hydrolysis reaction this is usually not the practice.
The enzymatic hydrolysis and alcoholysis of esters of 1,2-diol monotosylates is known. Unfortunately, the enzymatic esterification of 1,2-diol monotosylates under standard conditions, though reported, can suffer from severe complications. This is exemplified by the attempted enzymatic esterification of 1-tosyloxy-2-hydroxy-3-butene. The first significant problem is that the enzymatic esterification of this species often stops short of the optimal 50% conversion rate. In extreme cases, little conversion to products is observed at all. Only upon repeated addition of more enzyme does the reaction eventually reach 50% conversion. Further, the reaction products show only moderate enantiomeric excess. Improvement of the enzymatic esterification rate and percent conversion percentage as well as improvement of the enantiospecificity is necessary before enzymatic esterification of 1,2-diol monosulfonates is suitable for commercial scale.